A New Method to Detect Variants of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Bioluminescent Assay in Real Time (RT-LAMP-BART).

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), of which there are several variants. The three major variants (Alpha, Delta, and Omicron) carry the N501Y, L452R, and Q493R/Q498R mutations, respectively, in the S gene. Control of COVID-19 requires rapid and reliable detection of not only SARS-CoV-2 but also its variants. We previously developed a reverse transcription loop-mediated isothermal amplification assay combined with a bioluminescent assay in real time (RT-LAMP-BART) to detect the L452R mutation in the SARS-CoV-2 spike protein. In this study, we established LAMP primers and peptide nucleic acid probes to detect N501Y and Q493R/Q498R. The LAMP primer sets and PNA probes were designed for the N501Y and Q493R/Q498R mutations on the S gene of SARS-CoV-2. The specificities of RT-LAMP-BART assays were evaluated using five viral and four bacterial reference strains. The sensitivities of RT-LAMP-BART assays were evaluated using synthetic RNAs that included the target sequences, together with RNA-spiked clinical nasopharyngeal and salivary specimens. The results were compared with those of conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) methods. The method correctly identified N501Y and Q493R/Q498R. Within 30 min, the RT-LAMP-BART assays detected up to 100-200 copies of the target genes; conventional real-time RT-PCR required 130 min and detected up to 500-3000 copies. Surprisingly, the real-time RT-PCR for N501Y did not detect the BA.1 and BA.2 variants (Omicron) that exhibited the N501Y mutation. The novel RT-LAMP-BART assay is highly specific and more sensitive than conventional real-time RT-PCR. The new assay is simple, inexpensive, and rapid; thus, it can be useful in efforts to identify SARS-CoV-2 variants of concern.

Porphyromonas gingivalis hydrogen sulfide enhances methyl mercaptan-induced pathogenicity in mouse abscess formation.

Porphyromonas gingivalis produces hydrogen sulfide (H2S) from l-cysteine. However, the role of H2S produced by P. gingivalis in periodontal inflammation is unclear. In this study, we identified the enzyme that catalyses H2S production from l-cysteine and analysed the role of H2S using a mouse abscess model. The enzyme identified was identical to methionine γ-lyase (PG0343), which produces methyl mercaptan (CH3SH) from l-methionine. Therefore, we analysed H2S and CH3SH production by P. gingivalis W83 and a PG0343-deletion mutant (ΔPG0343) with/without l-cysteine and/or l-methionine. The results indicated that CH3SH is produced constitutively irrespective of the presence of l-methionine, while H2S was greatly increased by both P. gingivalis W83 and ΔPG0343 in the presence of l-cysteine. In contrast, CH3SH production by ΔPG0343 was absent irrespective of the presence of l-methionine, and H2S production was eliminated in the absence of l-cysteine. Thus, CH3SH and H2S production involves different substrates, l-methionine or l-cysteine, respectively. Based on these characteristics, we analysed the roles of CH3SH and H2S in abscess formation in mice by P. gingivalis W83 and ΔPG0343. Abscess formation by P. gingivalis W83, but not ΔPG0343, differed significantly in the presence and absence of l-cysteine. In addition, the presence of l-methionine did not affect the size of abscesses generated by P. gingivalis W83 and ΔPG0343. Therefore, we conclude that H2S produced by P. gingivalis does not induce inflammation; however, H2S enhances inflammation caused by CH3SH. Thus, these results suggest the H2S produced by P. gingivalis plays a supportive role in inflammation caused by methionine γ-lyase.

Involvement of PorK, a component of the type IX secretion system, in Prevotella melaninogenica pathogenicity.

Prevotella melaninogenica is a gram-negative anaerobic commensal bacterium that resides in the human oral cavity and is isolated as a pathogen of suppurative diseases both inside and outside the mouth. However, little is known about the pathogenic factors of P. melaninogenica. The periodontal pathogens Porphyromonas gingivalis and Tanerella forsythia secrete virulence factors such as protease and bacterial cell surface proteins via a type IX secretion system (T9SS) that are involved in pathogenicity. P. melaninogenica also possesses all known orthologs of T9SS. In this study, a P. melaninogenica GAI 07411 mutant deficient in the orthologue of the T9SS-encoding gene, porK, was constructed. Hemagglutination and biofilm formation were decreased in the porK mutant. Furthermore, following growth on skim milk-containing medium, the diameters of the halos surrounding the porK mutant were smaller than those of the wild-type strain, suggesting a decrease in secretion of proteases outside the bacterium. To investigate this in detail, culture supernatants of wild-type and porK mutant strains were purified and compared by two-dimensional electrophoresis. In the mutant strain, fewer spots were detected, indicating fewer secreted proteins. In infection experiments, the mortality rate of mice inoculated with the porK mutant strain was significantly lower than in the wild-type strain. These results suggest that P. melaninogenica secretes potent virulence factors via the T9SS that contribute to its pathogenic ability.

Involvement of an Skp-Like Protein, PGN_0300, in the Type IX Secretion System of Porphyromonas gingivalis.

The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence.

Acute oral complications in a pediatric patient with acute lymphoid leukemia.

Pseudomonas aeruginosa is a low-virulence pathogen that can cause infection in immunocompromised patients. Among pediatric hematologists, it is known that careful attention should be paid to P. aeruginosa as the bacteria responsible for intraoral inflammation, and antibiotic therapy targeting P. aeruginosa is the first-line treatment during neutropenia in patients with hematological malignancy. Immunosuppressed patients, however, are at high risk of developing inflammation. Here, we report a case involving a 10-year-old patient with acute lymphoid leukemia (ALL), in which P. aeruginosa synergistically acted with pre-existing gingivitis to induce unusual oral necrotic lesions, leading to acute buccal cellulitis and pus discharge from external sinus tracts.

Extracellular histones promote calcium phosphate-dependent calcification in mouse vascular smooth muscle cells.

Vascular calcification, a major risk factor for cardiovascular events, is associated with a poor prognosis in chronic kidney disease (CKD) patients. This process is often associated with the transformation of vascular smooth muscle cells (VSMCs) into cells with osteoblast-like characteristics. Damage-associated molecular patterns (DAMPs), such as extracellular histones released from damaged or dying cells, are suspected to accumulate at calcification sites. To investigate the potential involvement of DAMPs in vascular calcification, we assessed the impact of externally added histones (extracellular histones) on calcium and inorganic phosphate-induced calcification in mouse VSMCs. Our study found that extracellular histones intensified calcification. We also observed that the histones decreased the expression of VSMC marker genes while simultaneously increasing the expression of osteoblast marker genes. Additionally, histones treated with DNase I, which degrades dsDNA, attenuated this calcification, compared with the non-treated histones, suggesting a potential involvement of dsDNA in this process. Elevated levels of dsDNA were also detected in the serum of CKD model mice, underlining its potential role in vascular calcification in CKD. Our findings suggest that extracellular histones could play a pivotal role in the vascular calcification observed in CKD.

Suppression of Streptococcus mutans Biofilm Formation and Gene Expression by PRG Barrier Coat: A Molecular and Microscopic Study for Preventing Dental Caries.

PURPOSE: This study aimed to investigate the inhibitory effect of a PRG Barrier Coat on biofilm formation and structure by Streptococcus mutans and propose an effective method for preventing dental caries. MATERIALS AND METHODS: Streptococcus mutans MT8148 biofilms were obtained from hydroxyapatite disks with and with- out a PRG Barrier Coat. Scanning electron microscopy (SEM) was used to observe the 12- and 24-h-cultured biofilms, while reverse-transcription polymerase chain reaction (qRT-PCR) was used to quantify caries-related genes. Biofilm adhe- sion assessments were performed on glass. Statistical analysis was performed using a two-sample t-test. RESULTS: A statistically significant difference in Streptococcus mutans biofilm adhesion rate was observed between the con- trol and PRG Barrier Coat-coated samples (p < 0.01). However, there was no statistically significant difference in total bacter- ial count or biofilm volume (p > 0.05). SEM revealed that the PRG Barrier Coat inhibited biofilm formation by Streptococcus mutans. Real-time RT-PCR revealed that the material restricted the expression of genes associated with caries-related bio- film formation. However, the suppression of gtfD and dexB differed from that of other genes. CONCLUSION: PRG Barrier Coat suppressed biofilm formation by Streptococcus mutans by inhibiting the expression of in- soluble glucan synthase, which is associated with primary biofilm formation. The material also affected gene expression and altered the biofilm structure. Tooth surface-coating materials, such as PRG Barrier Coat, may improve caries preven- tion in dental practice.

The expression of MIR125B transcripts and bone phenotypes in Mir125b2-deficient mice.

MIR125B, particularly its 5p strand, is apparently involved in multiple cellular processes, including osteoblastogenesis and osteoclastogenesis. Given that MIR125B is transcribed from the loci Mir125b1 and Mir125b2, three mature transcripts (MIR125B-5p, MIR125B1-3p, and MIR125B2-3p) are generated (MIR125B-5p is common to both); however, their expression profiles and roles in the bones remain poorly understood. Both primary and mature MIR125B transcripts were differentially expressed in various organs, tissues, and cells, and their expression patterns did not necessarily correlate in wild-type (WT) mice. We generated Mir125b2 knockout (KO) mice to examine the contribution of Mir125b2 to MIR125B expression profiles and bone phenotypes. Mir125b2 KO mice were born and grew normally without any changes in bone parameters. Interestingly, in WT and Mir125b2 KO, MIR125B-5p was abundant in the calvaria and bone marrow stromal cells. These results indicate that the genetic ablation of Mir125b2 does not impinge on the bones of mice, attracting greater attention to MIR125B-5p derived from Mir125b1. Future studies should investigate the conditional deletion of Mir125b1 and both Mir125b1 and Mir125b2 in mice.

Transcriptomic Profiling Reveals Neuroinflammation in the Corpus Callosum of a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a widespread neurodegenerative disorder characterized by progressive cognitive decline, affecting a significant portion of the aging population. While the cerebral cortex and hippocampus have been the primary focus of AD research, accumulating evidence suggests that white matter lesions in the brain, particularly in the corpus callosum, play an important role in the pathogenesis of the disease. OBJECTIVE: This study aims to investigate the gene expression changes in the corpus callosum of 5xFAD transgenic mice, a widely used AD mouse model. METHODS: We conducted behavioral tests for spatial learning and memory in 5xFAD transgenic mice and performed RNA sequencing analyses on the corpus callosum to examine transcriptomic changes. RESULTS: Our results show cognitive decline and demyelination in the corpus callosum of 5xFAD transgenic mice. Transcriptomic analysis reveals a predominance of upregulated genes in AD mice, particularly those associated with immune cells, including microglia. Conversely, downregulation of genes related to chaperone function and clock genes such as Per1, Per2, and Cry1 is also observed. CONCLUSIONS: This study suggests that activation of neuroinflammation, disruption of chaperone function, and circadian dysfunction are involved in the pathogenesis of white matter lesions in AD. The findings provide insights into potential therapeutic targets and highlight the importance of addressing white matter pathology and circadian dysfunction in AD treatment strategies.

SARS-CoV-2 detection in pediatric dental clinic wastewater reflects the number of local COVID-19 cases in children under 10 years old.

This was the first longitudinal study to analyze dental clinic wastewater to estimate asymptomatic SARS-CoV-2 infection trends in children. We monitored wastewater over a 14-month period, spanning three major COVID-19 waves driven by the Alpha, Delta, and Omicron variants. Each Saturday, wastewater was sampled at the Pediatric Dental Clinic of the only dental hospital in Japan's Saitama Prefecture. The relationship between the weekly number of cases in Saitama Prefecture among residents aged < 10 years (exposure) and wastewater SARS-CoV-2 RNA detection (outcome) was examined. The number of cases was significantly associated with wastewater SARS-CoV-2 RNA positivity (risk ratio, 5.36; 95% confidence interval, 1.72-16.67; Fisher's exact test, p = 0.0005). A sample from Week 8 of 2022 harbored the Omicron variant. Compared to sporadic individual testing, this approach allows continuous population-level surveillance, which is less affected by healthcare seeking and test availability. Since wastewater from pediatric dental clinics originates from the oral cavities of asymptomatic children, such testing can provide important information regarding asymptomatic COVID-19 in children, complementing clinical pediatric data.

Taxonomy of the Anginosus group of the genus Streptococcus and description of Streptococcus anginosus subsp. whileyi subsp. nov. and Streptococcus constellatus subsp. viborgensis subsp. nov.

The Anginosus group of the genus Streptococcus has been the subject of much taxonomic confusion, which has hampered the full appreciation of its clinical significance. The purpose of this study was to critically re-examine the taxonomy of the Anginosus group, with special attention to ß-haemolytic, Lancefield group C strains, using multilocus sequence analysis (MLSA) combined with 16S rRNA gene sequence and phenotypic analyses. Phylogenetic analysis of concatenated sequences of seven housekeeping genes previously used for examination of viridans streptococci distinguished seven distinct and coherent clusters in the Anginosus group. Analyses of 16S rRNA gene sequences and phenotypic characters supported the MLSA clustering and currently recognized taxa of the Anginosus group. Single gene analyses showed considerable allele sharing between species, thereby invalidating identification based on single-locus sequencing. Two novel clusters of ß-haemolytic, Lancefield group C strains within the Streptococcus constellatus and Streptococcus anginosus species and isolated from patients with sore throat showed sufficient phylogenetic distances from other clusters to warrant status as novel subspecies. The novel cluster within S. anginosus was identified as the previously recognized DNA homology cluster, DNA group 2. The names S. anginosus subsp. whileyi subsp. nov. (type strain CCUG 39159(T) = DSM 25818(T) = SK1267(T)) and S. constellatus subsp. viborgensis subsp. nov. (type strain SK1359(T) = CCUG 62387(T) = DSM 25819(T)) are proposed.

Management of maxillary alveolar bone fracture and severely intruded maxillary central incisor: report of a case.

An 11-year-old male who injured his maxilla and right maxillary central incisor and lip during a fall was presented to our hospital. His lower lip and upper gingiva were lacerated with swelling and epistaxis, and he had a maxillary alveolar bone fracture and severe intrusion of the right maxillary central incisor, which had penetrated the floor of the nasal cavity with avulsion. Under local anesthesia, we repositioned the incisor and bone segment and fixed them with a titanium micromesh plate and self-tapping screws and splints. The incisor was also treated by root canal 3 days after the operation and was restored with a crown. We performed root canal filling 1 month later. Five months later, the plate and screws were removed. In prognosis of our case, no symptoms of inflammatory root resorption or ankylosis have observed for more than 1 year and 6 months of follow up based on both clinical and radiographic findings.

Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis.

BACKGROUND: Aggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530) in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone. METHODS: Based on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97-100%) among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer-probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis. RESULTS: The JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 10(8) (mean 1.28 × 10(7)) for JP2 clones and from 0 to 1.6 × 10(6) for non-JP2 clones (mean 1.84 × 10(5)). There were significant differences in the JP2 cell number between a clinical attachment level (CAL) ≤6 mm and a level ≥7 mm (p < 0.01). Our new qPCR-based JP2- and non-JP2-specific quantitative detection assay is applicable to the diagnosis of aggressive periodontitis with A. actinomycetemcomitans. CONCLUSIONS: We successfully developed a quantitative and discriminative PCR-based method for the detection of A. actinomycetemcomitans JP2 and non-JP2 clones. This technique will contribute to future analyses of the quantitative relationship between this organism and aggressive periodontitis.

Establishment of Streptococcus mutans in infants induces decrease in the proportion of salivary α-haemolytic bacteria.

OBJECTIVE: For paediatric dentists, an indicator to assess caries risk of infants is very important. Conventionally, the number and/or proportions of Streptococcus mutans have been employed as risk indicator; however, because such figures reflect the existing situation, they are not suitable for assessing caries risk of infants that have not yet been infected with S. mutans. Thus, we searched for an indicator for the establishment of S. mutans. METHODS: To evaluate the changes caused by the establishment of S. mutans in the microbiota of the infant oral cavity, we monitored changes in the oral microbiota of two pre-dentate infants over a 3-year period and in a cross-sectional study of 40 nursery school-aged children by cultivation of saliva on nonselective blood agar, Mitis-Salivarius agar, and Mitis-Salivarius agar supplemented with bacitracin combined with identification of selected isolates. RESULTS: Two longitudinal observations suggested that the establishment of S. mutans would induce a decrease in α-haemolytic bacteria in the microbial population of the oral cavity. This suggestion was compensated with the results of cross-sectional study, and it was revealed that the establishment of 10(3) CFU/mL of mutans streptococci in saliva might be predicted by a microbiota comprising less than approximately 55% of α-haemolytic. CONCLUSION: Decrease in the proportion of α-haemolytic bacteria in saliva of infant was found to be applicable as an indicator to predict the establishment of S. mutans and to assess dental caries risk as a background for planning of dental care and treatment in the infants before infection with S. mutans.

Comparison of oral streptococci biofilm in caries-free and caries-affected preschool Mexican children.

Interaction of oral streptococci biofilm is the main etiological factor for dental caries. The aim of the study was to compare oral streptococci (OS) distribution in the biofilm of primary dentition from caries-free and caries-affected preschool Mexican children. This cross-sectional study involved 40 caries-free and 40 caries-affected children with primary dentition. Each child was examined using the dmfs index, DNA was extracted from saliva and presence of OS was determined by PCR. Data obtained showed no statistical difference regarding age and gender (P > 0.05). Streptococcus mutans (Smut), Streptococcus sobrinus (Ssob) and their combination showed significant statistical differences between groups (P < 0.05). Smut, Streptococcus sanguinis and Streptococcus gordonii had an inverse relation with dmfs index and Ssob had a direct relation similar to combined with Smut. Smut-Ssob combined with other OS showed statistical differences (P < 0.05). In free-caries group Streptococcus gordonii was more frequently identified than Smut. The ratio Smut/Streptococcus sanguinis could represent a high risk of dental caries development; this ratio was higher in the caries-affected (1.18) than in the caries-free group (0.32). In conclusion, OS play an important role in dental caries predisposition and severity, not only the presence of Smut and Srob, but also the complexity and distribution of OS in the biofilm.

The findings of glucosyltransferase enzymes derived from oral streptococci.

Glucosyltransferase enzymes (Gtfs) distribute among some streptococcal species in oral cavity and are known as key enzymes contributing to the development of oral biofilm such as dental plaque. In 18 streptococcal species, 45 glucosyltransferase genes (gtf) are detected from genome database. Gtfs catalyze the synthesis of the glucans, which are polymers of glucose, from sucrose and they are main component of oral biofilm. Especially, the Gtfs from Streptococcus mutans are recognized as one of dental caries pathogens since they contribute to the formation of dental plaque and the establishment of S. mutans in the tooth surface. Therefore, Gtfs has been studied particularly by many researchers in the dentistry field to develop the anti- caries vaccine. However, it is not still accomplished. In these days, the phylogenetic and crystal structure analyses of Gtfs were performed and the study of Gtfs will enter new situation from the technique in the past old viewpoint. The findings from those analyses will affect the development of the anti-caries vaccine very much after this.　In this review, we summarize the findings of oral streptococcal Gtfs and consider the perspectives of the dental caries prevention which targeted Gtf.

Detection of SARS-CoV-2 and the L452R spike mutation using reverse transcription loop-mediated isothermal amplification plus bioluminescent assay in real-time (RT-LAMP-BART).

The new coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be fatal, and several variants of SARS-CoV-2 with mutations of the receptor-binding domain (RBD) have increased avidity for human cell receptors. A single missense mutation of U to G at nucleotide position 1355 (U1355G) in the spike (S) gene changes leucine to arginine (L452R) in the spike protein. This mutation has been observed in the India and California strains (B.1.617 and B.1.427/B.1.429, respectively). Control of COVID-19 requires rapid and reliable detection of SARS-CoV-2. Therefore, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay plus a bioluminescent assay in real-time (BART) to detect SARS-CoV-2 and the L452R spike mutation. The specificity and sensitivity of the RT-LAMP-BART assay was evaluated using synthetic RNAs including target sequences and RNA-spiked clinical nasopharyngeal and saliva specimens as well as reference strains representing five viral and four bacterial pathogens. The novel RT-LAMP-BART assay to detect SARS-CoV-2 was highly specific compared to the conventional real-time RT-PCR. Within 25 min, the RT-LAMP-BART assay detected 80 copies of the target gene in a sample, whereas the conventional real-time RT-PCR method detected 5 copies per reaction within 130 min. Using RNA-spiked specimens, the sensitivity of the RT-LAMP-BART assay was slightly attenuated compared to purified RNA as a template. The results were identical to those of the conventional real-time RT-PCR method. Furthermore, using a peptide nucleic acid (PNA) probe, the RT-LAMP-BART method correctly identified the L452R spike mutation. This is the first report describes RT-LAMP-BART as a simple, inexpensive, rapid, and useful assay for detection of SARS-CoV-2, its variants of concern, and for screening of COVID-19.

Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria.

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-ß-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four ß-lactamase genes (bla KPC, bla NDM-1, bla IMP-1 group, and bla VIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six ß-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four ß-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four ß-lactamases.

6-Deoxyjacareubin, a natural compound preventing hypoxia-induced cell death, ameliorates neurodegeneration in a mouse model of familial amyotrophic lateral sclerosis.

The central nervous system (CNS) uses a significant amount of oxygen for energy production. Decreased oxygen supply due to impaired blood supply critically damages the CNS. As chronic hypoxic conditions have diverse effects via the excessive production of reactive oxygen species, protection from hypoxic damage is important for cell survival. Recent studies have revealed that various markers of hypoxia are altered in age-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), indicating the involvement of hypoxia. However, therapeutic strategies targeting hypoxia-induced pathways in ALS have not been developed yet. We previously screened small-molecule compounds that inhibit hypoxia-induced cell death and identified 6-deoxyjacareubin. We hypothesized that the modulation of hypoxia signaling by 6-deoxyjacareubin might protect motor neurons in ALS. Here, we show that 6-deoxyjacareubin indeed ameliorates neurodegeneration in a mouse model of familial ALS. Administration of 6-deoxyjacareubin to this familial ALS model significantly attenuated disease progression and improved locomotor dysfunction. We also found that 6-deoxyjacareubin reduced motor neuron loss and glial activation. Our results indicate that 6-deoxyjacareubin might serve as a potential therapeutic tool for ALS. Moreover, these results suggest that modulation of hypoxia signaling pathways provides a promising strategy to develop therapies for other types of neurodegenerative diseases also characterized by hypoxia.

Apparent diet digestibility of captive colobines in relation to stomach types with special reference to fibre digestion.

Colobine monkeys are known for the anatomical complexity of their stomachs, making them distinct within the primate order. Amongst foregut fermenters, they appear peculiar because of the occurrence of two different stomach types, having either three ('tripartite') or four ('quadripartite', adding the praesaccus) chambers. The functional differences between tri and quadripartite stomachs largely remain to be explained. In this study, we aim to compare the apparent digestibility (aD) in tripartite and quadripartite colobines. Hence, we measured the aD in two colobine species, Nasalis larvatus (quadripartite) and Trachypithecus cristatus (tripartite), in two zoos. We also included existing colobine literature data on the aD and analysed whether the aD of fibre components is different between the stomach types to test the hypothesis of whether quadripartite colobines show higher aD of fibre components than tripartite colobines did. Our captive N. larvatus specimen had a more distinctively varying nutrient intake across seasons with a larger seasonal variation in aD than that of a pair of T. cristatus, which mostly consumed commercial foods with a lower proportion of browse and less seasonal variation. We observed higher aD of dry matter (DM), neutral detergent fibre (NDF) and acid detergent fibre (ADF) in the N. larvatus specimen, suggesting a higher gut capacity of N. larvatus provided by the additional praesaccus forestomach chamber. Based on the analysis of literature data for aD, we also found that quadripartite species achieved higher fibre digestibility at similar dietary fibre levels compared with tripartite species, supporting the hypothesis that the additional gut capacity offered by the praesaccus facilitates a longer retention and hence more thorough microbial fermentation of plant fibre.